The mechanism of the energy linked transhydrogenase reaction is being studied by the application of a technique of resolution, purification and reconstitution of this activity with a soluble transhydrogenase factor and R. rubrum membrane fractions. The reconstituted system can be activated by light, adenosine triphosphate or pyrophosphate and as such provides as well an ideal system for the investigation of the general characteristics of energy conservation systems. A fluorometric assay for the transhydrogenase reaction makes such an analysis extremely sensitive. The ability to prepare membrane fractions totally resolved with respect to first, adenosine triphosphatase and then pyrophosphatase activity (and thus unable to photophosphorylate adenosine diphosphate or inorganic phosphate) provides membrane particles in which the energized intermediate(s) generated by light by adenosine triphosphate and by pyrophosphate can be at least operationally separated from each other and analyzed as separate entities. In addition the mechanism of pyrophosphate synthesis is being investigated by means of a pyrophosphate trapping system and a specific fluorometric-enzymatic analysis of the products of this trap. It is anticipated that the reconstituted transhydrogenase activity will be used to study 1) the mechanism of the reaction itself, 2) the combination of a specific soluble protein (without any enzymatic activity) with a membrane particulate (also without transhydrogenase activity) which when combined catalyzes a specific enzymatic reaction, 3) the formation of the energized intermediates produced separately by light, adenosine triphosphate and pyrophosphate and finally, 4) the mechanism of photopyrophosphorylation compared with photophosphorylation of adenosine diphosphate. The general approach being used in the laboratory should enable us to arrive at a definition of the energized intermediates (states) postulated for bacterial (and mitochondrial) energy conservation systems. Our studies will define as well the characteristic features of the interaction of a soluble protein with a membrane particulate.